Genome Sequence of Lactococcus garvieae IPLA 31405, a Bacteriocin-Producing, Tetracycline-Resistant Strain Isolated from a Raw-Milk Cheese

This work describes the draft genome sequence of Lactococcus garvieae IPLA 31405, isolated from a traditional Spanish cheese. The genome contains a lactose-galactose operon, a bacteriocin locus, two integrated phages, a transposon harboring an active tet(M) gene, and two theta-type plasmid replicons. Genes encoding virulence factors were not recorded.     

The Plasmid Complement of Lactococcus lactis UC509.9 Encodes Multiple Bacteriophage Resistance Systems

Lactococcus lactis subsp. cremoris strains are used globally for the production of fermented dairy products, particularly hard cheeses. Believed to be of plant origin, L. lactis strains that are used as starter cultures have undergone extensive adaptation to the dairy environment, partially through the acquisition of extrachromosomal DNA in the form of plasmids that specify technologically important phenotypic traits. Here, we present a detailed analysis of the eight plasmids of L. lactis UC509.9, an Irish dairy starter strain. Key industrial phenotypes were mapped, and genes that are typically associated with lactococcal plasmids were identified. Four distinct, plasmid-borne bacteriophage resistance systems were identified, including two abortive infection systems, AbiB and AbiD1, thereby supporting the observed phage resistance of L. lactis UC509.9. AbiB escape mutants were generated for phage sk1, which were found to carry mutations in orf6, which encodes the major capsid protein of this phage.     

Genome Sequence of the Bacteriocin-Producing Strain Lactococcus garvieae DCC43

This work describes the draft genome sequence of Lactococcus garvieae DCC43. The 2.2-Mb draft genome contains 2,227 predicted protein-coding genes, among which is a region encoding the bacteriocin garvicin ML. No antibiotic resistance genes or capsule-related virulence genes were identified. Two plasmid replication regions indicate that this strain likely contains plasmids. Comparative genomics suggests that this strain displays a high degree of sequence variation from the previously sequenced L. garvieae strains.     

The protective immune response of yellowtail Seriola quinqueradiata to the bacterial fish pathogen Lactococcus garvieae.

Yellowtail Seriola quinqueradiata were immunized with 2 different Lactococcus garvieae bacterin, formalin-killed KG- phenotype cells (capsulated phenotype) and formalin-killed KG+ phenotype cells (unencapsulated phenotype). These 2 injected vaccines conferred long-term protection to yellowtail against an artificial infection of an encapsulated Lactococcus garvieae strain with long-lasting agglutinating titres against KG+ phenotype cells. However, no agglutinating titres or low agglutinating titres against KG- phenotype cells were detected in fish given each of these bacterin. These results suggested that a capsule in KG- phenotype cells apparently affects their immunogenicity, but the antigens which conferred protection to fish against lactococcal infection may be located on the surface of KG+ phenotype cells, and are not cell capsules in KG- phenotype cells. The protection offered by a formalin-killed KG+ phenotype cell vaccine would not appear to be strain specific. Encapsulated L. garvieae cells were well phagocytosed, and fimbrie-like appendages were seen in KG- phenotype cells after treatment with yellowtail immune serum.     

Clonality and Diversity of the Fish Pathogen Lactococcus garvieae in Mediterranean Countries

Infection with Lactococcus garvieae is considered the most important risk factor for the European trout industry, and the losses are approximately 50% of the total production. To improve our understanding of the genetic links among strains originating from different countries, we examined the population structure of L. garvieae by comparing 81 strains isolated from different sources and ecosystems (41 farms in six countries) in which the bacterium is commonly found. Genetic similarities (as assessed with molecular tools, including restriction fragment length polymorphism ribotyping with two endonucleases) were compared with serological data. The combined results reveal that in endemic sites the bacterial population displays a clonal structure, whereas bacterial diversity characterizes sites where the infection is sporadic.     

Complete genome sequence and comparative analysis of the fish pathogen Lactococcus garvieae

Lactococcus garvieae causes fatal haemorrhagic septicaemia in fish such as yellowtail. The comparative analysis of genomes of a virulent strain Lg2 and a non-virulent strain ATCC 49156 of L. garvieae revealed that the two strains shared a high degree of sequence identity, but Lg2 had a 16.5-kb capsule gene cluster that is absent in ATCC 49156. The capsule gene cluster was composed of 15 genes, of which eight genes are highly conserved with those in exopolysaccharide biosynthesis gene cluster often found in Lactococcus lactis strains. Sequence analysis of the capsule gene cluster in the less virulent strain L. garvieae Lg2-S, Lg2-derived strain, showed that two conserved genes were disrupted by a single base pair deletion, respectively. These results strongly suggest that the capsule is crucial for virulence of Lg2. The capsule gene cluster of Lg2 may be a genomic island from several features such as the presence of insertion sequences flanked on both ends, different GC content from the chromosomal average, integration into the locus syntenic to other lactococcal genome sequences, and distribution in human gut microbiomes. The analysis also predicted other potential virulence factors such as haemolysin. The present study provides new insights into understanding of the virulence mechanisms of L. garvieae in fish.     

Biochemical Adaptation to the Environment

SYNOPSIS. Biochemical adaptation to environmental parameterssuch as temperature appears to involve two distinct types ofchanges in the organism's chemistry. On the one hand, the quantitiesof certain molecular species present in the cells may change.Alternatively, the actual types of molecules present may vary.Rainbow trout (Salmo gairdneri) acclimated to warm and coldtemperatures exhibit a striking example of this latter typeof adaptation. For all enzymes we have examined in this species,distinct "warm" and "cold" isozymes are present. The isozymesfound in warmacclimated (18°C) trout function well onlyat temperatures above 10–12°C. The isozymes presentin cold-acclimated (4°C) trout function optimally at 2-5°C,temperatures this species normally encounters in winter. Thesedata, plus information on comparable changes in membrane lipids,lead us to propose that adult poikilotherms may undergo a considerabledegree of "biochemical restructuring" on a seasonal basis. Thefactors which control this "restructuring," and the rates atwhich the process occurs at high and low temperatures, are topicsfor future investigation.     

The susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae and its resistance under copper sulfate stress.

Addition of copper sulfate (0.1 to 0.4 mg l(-1)) to tryptic soy broth (TSB) had no effect on growth rate of the bacterial pathogen Lactococcus garvieae. Giant freshwater prawns Macrobrachium rosenbergii were injected with L. garvieae (4 x 10(6) colony-forming units [cfu] prawn(-1)) grown in TSB or TSB containing copper sulfate at 0.1, 0.2, 0.3 or 0.4 mg l(-1). After 48 h, the cumulative mortality was significantly (p < 0.05) higher for prawns exposed to L. garvieae grown in 0.4 mg l(-1) copper sulfate than at the lower concentrations examined. In other experiments, prawns were injected with TSB-grown L. garvieae (4 x 10(6) and 2 x 10(5) cfu prawn(-1)), then held in water containing copper sulfate. After 8 h the mortality of L. garvieae-exposed prawns held in water containing 0.4 mg l(-1) copper sulfate was significantly higher than prawns held in water containing 0.2 and 0.3 mg l(-1) copper sulfate. At the lower L. garvieae density, cumulative mortality of prawns increased directly with ambient copper sulfate concentrations in the range of 0.2 to 0.4 mg l(-1). All prawns survived a 168 h exposure to 0.1 mg l(-1) copper sulfate. Prawns exposed to different concentrations of copper sulfate were examined for hemocyte density, phenoloxidase activity and respiratory burst. No significant differences in hemocyte density were observed among treatments. In prawns following a 48 h exposure to 0.1 mg l(-1) copper sulfate, phenoloxidase activity was decreased, but respiratory burst was increased. In conclusion, copper sulfate increased the virulence of L. garvieae to M. rosenbergii and modulated its immune system. Copper sulfate at 0.1 mg l(-1) decreased susceptibility of M. rosenbergii to L garvieae infection, whereas at 0.2 mg l(-1) the susceptibility was increased. The generation of superoxide anion by M. rosenbergii exposed to copper sulfate at a concentration higher than 0.2 mg l(-1) was considered to be cytoxic.     

Isolation of Lactococcus garvieae Strain TRF1 from the Fecal Material of a Timber Rattlesnake

In certain species of fish, such as rainbow trout, infection by the Firmicutes Lactococcus garvieae is problematic. This organism is the causative agent of lactococcosis disease in fish, and it is also considered a potential zoonotic bacterium, since it can cause several opportunistic infections in humans. In this study, L. garvieae strain TRF1 was grown and isolated in pure culture from the fecal material of a Timber Rattlesnake (Crotalus horridus), living in the wild. The presence/absence of several putative virulence factors was identified using staining, PCR amplification, and the construction of a draft genome. Strain TRF1 shared several putative virulence factors with strain Lg2, a known fish pathogen. However, the capsule gene cluster, found in strain Lg2, was not found in strain TRF1. Since this gene cluster is absent in several non-pathogenic strains of L. garvieae, it suggests strain TRF1 may not be pathogenic. However, this hypothesis will have to be tested in an animal model.     

Cell-surface properties of Lactococcus garvieae strains and their immunogenicity in the yellowtail Seriola quinqueradiata

The cell-surface properties of strains of Lactococcus garvieae were examined. Two capsular types were found, one with a highly developed capsule (KG9408) and one with a micro-capsule (MS93003) carrying fimbriae-like components projecting from the cell surface. One strain (NSS9310) had neither cell capsular nor fimbriae-like structures on its cell surface. The strains with the highly developed capsule were more virulent to fish than either the micro-capsular or non-capsular strains. The KG9408, MS93003 and NSS9310 strains could be clearly differentiated by their susceptibility to bacteriophages. Protection against L. garvieae infection was induced in the yellowtail Seriola quinqueradiata by immunization with formalin-killed L. garvieae KG9408 and MS93003 cells. Although protection was also induced by immunization with NSS9310, the level of protection was significantly lower than that with KG9408 and MS93003 vaccines. Passive immunization with yellowtail immune sera raised against KG9408 and MS93003 conferred strong protection on yellowtail with rapid bacterial clearance after challenge with L. garvieae. Immunoblotting analysis of protein antigens extracted from L. garvieae strains using rabbit anti-KG9408 and anti-MS93003 sera and yellowtail anti-KG9408 and anti-MS93003 sera indicated that some bands in KG9408 and MS93003 strains were not detectable in NSS9310.     

Dopamine depresses the immune ability and increases susceptibility to Lactococcus garvieae in the freshwater giant prawn, Macrobrachium rosenbergii

The total haemocyte count (THC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (16.2 +/- 2.1 g) were individually injected with saline, or dopamine at 0.5, 5.0, or 50.0 pmol prawn(-1). The results show that a transient period of immunosuppression occurred between 2 and 8 h after injection of dopamine for all immune parameters except circulating haemocytes and all immune parameters returned to control values within 8-16 h after receiving dopamine. Injection of dopamine also significantly increased the mortality of M. rosenbergii challenged with the pathogen L. garvieae. These results suggest that stress-inducing dopamine suppresses the immune system, which in turn promotes the susceptibility to L. garvieae in M. rosenbergii.     

Effect of benzalkonium chloride stress on immune resistance and susceptibility to Lactococcus garvieae in the giant freshwater prawn Macrobrachium rosenbergii

Addition of benzalkonium chloride (BKC) at 0, 0.3, 0.6 and 1.0 mg l(-1) to tryptic soy broth (TSB) had no effect on growth of Lactococcus garvieae, a bacterial pathogen of the giant freshwater prawn Macrobrachium rosenbergii. However, injection of the cultured cells into prawns at a dose of 4 x 10(6) colony-forming units (cfu) prawn(-1) resulted in significantly higher mortality at 120 h (p < 0.05) in prawns injected with cells grown in the absence of BKC than in prawns injected with cells grown in the presence of BKC. In other experiments, prawns were injected with TSB-grown L. garvieae (4 x 10(6) and 3 x 10(5) cfu prawn(-1)) and then held in water containing BKC at 0, 0.3, 0.6 and 1.0 mg l(-1). After 120 h, mortality was significantly higher in all the BKC treatments than in the control without BKC. Prawns showed no significant differences in total hemocyte count (THC) or differential hemocyte count (DHC) amongst treatment and control groups. However, 96 h exposure to 0.3 mg l(-1) BKC or more resulted in a decrease in phenoloxidase activity and an increase in respiratory burst to levels considered to be cytoxic. In summary, exposure of L. garvieae to BKC at 0.3 mg l(-1) or more decreased its virulence to M. rosenbergii, while exposure of M. rosenbergii to BKC at 0.3 mg l(-1) or more increased its susceptibility to L. garvieae infection.     

Effect of nitrite on interaction between the giant freshwater prawn Macrobrachium rosenbergii and its pathogen Lactococcus garvieae

Addition of nitrite-N at 1.5 mg l(-1) in tryptic soy broth (TSB) significantly (p < 0.05) decreased the growth rate of the bacterial pathogen Lactococcus garvieae and significantly (p < 0.05) reduced mortality compared to zero nitrite controls when injected into giant freshwater prawns Macrobrachium rosenbergii at 5 x 10(5) colony-forming units (CFU) per prawn. In other experiments, whereby prawns were injected with TSB-grown L. garvieae (5 x 10(5) CFU prawn(-1)) and then held in water containing nitrite-N, mortality at 72 h post-injection was significantly (p < 0.05) higher for prawns held in water containing 1.68 mg l(-1) nitrite than at lower concentrations. Prawns exposed to different concentrations of nitrite-N were examined for THC (total hemocyte count), phenoloxidase activity, respiratory burst, phagocytic activity and bacterial clearance efficiency. No significant differences in THC and phenoloxidase activity were observed among treatments. With prawns exposed to nitrite-N for 168 h (7 d) at 1.59 mg l(-1), phagocytic activity and clearance efficiency decreased, while at 1.15 mg l(-1) or more, respiratory burst increased, generating the superoxide anion at levels considered cytoxic to the host. We conclude that nitrite-N at 1.68 mg l(-1) causes depression in the immune response and increased mortality in M. rosenbergii infected with L. garvieae.     

Effect of potassium permanganate stress on immune resistance and susceptibility to Lactococcus garvieae in the giant freshwater prawn Macrobrachium rosenbergii

This work is part of a continuing series of investigations on the effect of commonly used aquaculture chemicals on the immune resistance and susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae. The methodology has been described in earlier publications of the series. Potassium permanganate at 1.0 mg l(-1) in tryptic soy broth (TSB) had no effect on the growth rate of L. garvieae. The mortality of M. rosenbergii challenged with 4 x 10(6) colony-forming units (cfu) prawn(-1) of TSB-grown L. garvieae was significantly greater than that of challenged controls. Addition of potassium permanganate at 1.0 mg l(-1) in TSB significantly increased the virulence of L. garvieae to M. rosenbergii. Exposure of M. rosenbergii to potassium permanganate prior to challenge with TSB-grown L. garvieae at 4 x 10(6) and 3 x 10(6) cfu prawn(-1) revealed that 96 h mortality was significantly lower for prawns held in water containing 0.3 mg l(-1) of the chemical than for prawns in water containing 1.0 mg l(-1) or no chemical. Potassium permanganate caused no significant changes in total hemocyte counts and differential hemocyte counts, compared to the control treatments. However, a concentration of 1.0 mg l(-1) or more for 96 h resulted in decreased phenoloxidase activity, phagocytic activity and clearance efficiency. Respiratory burst increased with exposure to 0.3 mg l(-1). In conclusion, treatment with potassium permanganate at 0.3 mg 1(-1) was effective in reducing M. rosenbergii mortality from L. garvieae infection, but higher concentrations had a negative effect, probably due to reduced prawn defenses.     

Trichlorfon, an organophosphorus insecticide, depresses the immune responses and resistance to Lactococcus garvieae of the giant freshwater prawn Macrobrachium rosenbergii

The total haemocyte count (THC), phenoloxidase activity, respiratory bursts (release of superoxide anions), superoxide dismutase (SOD) activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (18.2+/-2.1 g) were individually reared in water containing concentrations of trichlorfon of 0, 0.2, or 0.4 mg L(-1) for a 144-h period. No significant differences in the THC were observed among prawns at the beginning and following 144 h of exposure to 0, 0.2, and 0.4 mg L(-1) trichlorfon. However, phenoloxidase activity significantly decreased when the prawns were exposed to trichlorfon at 0.2 and 0.4 mg L(-1), and no significant differences were observed between the two concentrations at any sampling time. The total production of superoxide anion by M. rosenbergii significantly increased with exposure to 0.2 and 0.4 mg L(-1) trichlorfon, and no significant differences were observed between the two concentrations during the 144-h exposure period. However, M. rosenbergii exposed to 0.2 and 0.4 mg L(-1) trichlorfon showed decreased SOD activity from 48 to 144 h. Phagocytic activity and clearance efficiency to L. garvieae significantly decreased when prawns were exposed to 0.2 and 0.4 mg L(-1) trichlorfon for 48 h. In another experiment, prawns were challenged with 5 x 10(5) colony-forming units (cfu)prawn(-1) of L. garvieae, then reared in water containing different concentrations of trichlorfon. The onset of mortality was earlier in prawns exposed to trichlorfon compared to those exposed to the zero control. The cumulative mortality of prawns directly increased with ambient trichlorfon concentrations in the range of 0-0.4 mg L(-1) after 168 h. It is concluded that exposure of M. rosenbergii to trichlorfon at 0.2 mg L(-1) or more caused cytotoxicity resulting in depression of the immune response, and increased its susceptibility to L. garvieae infection.     

Toxic Effects of Lactococcus garvieae,Staphylococcus epidermidis,and Bacillus subtilis Bacteria on the Physiology of Rainbow Trout

Biology Bulletin - In the trout production establishments all over the world, the most common pathogenic bacterial factors are increasing. Therefore, in this study, rainbow trout (Oncorhynchus...     

The Mode of Replication Is a Major Factor in Segregational Plasmid Instability in Lactococcus lactis

The effects of the rolling-circle and theta modes of replication on the maintenance of recombinant plasmids in Lactococcus lactis were studied. Heterologous Escherichia coli or bacteriophage λ DNA fragments of various sizes were inserted into vectors based on either the rolling-circle-type plasmid pWV01 or the theta-type plasmid pAMβ1. All pAMβ1 derivatives were stably maintained. pWV01 derivatives, however, showed size-dependent segregational instability, in particular when large DNA fragments were inserted. All recombinant pWV01 derivatives generated high-molecular-weight plasmid multimers (HMW) in amounts that were positively correlated with plasmid size and inversely correlated with the copy numbers of the monomeric plasmid forms. Formation of HMW or reductions in copy numbers were not observed with pAMβ1 derivatives. The results indicate that HMW formation and/or reduction in plasmid copy numbers is an important factor in the maintenance of pWV01 derivatives. It is concluded that theta-type plasmids are superior to rolling-circle-type plasmids for cloning in lactococci.